The invention relates in general to an apparatus and method for achieving electrophoretic focusing, and in particular to an apparatus for achieving electrophoretic separation and purification which is characterized by a separation chamber formed between two precision-pore insulated screens and which also includes inlet and outlet ports, a plurality of purge chambers for extracting extraneous fractions and for providing thermal cooling, a plurality of electrode chambers to provide a transverse electric field in the separation chamber, and pumping means for pumping sample, carrier buffer and electrode rinse buffer through the apparatus, and a method of employing this apparatus to achieve separation and collection of a desired component from a biological or chemical sample.
There are two electrokinetic methods that have had success separating biological materials, namely, zone electrophoresis and isoelectric focusing. Electrophoresis is the movement of suspended or dissolved charged particles in response to an applied electric field. The rate of motion depends upon the charge, size and shape of the particles and specific properties of the solvent buffer and its container. In zone electrophoresis, the components in a short sample zone are separated by the action of the electric field. The injection of a narrow, uniform zone and the absence of dispersive fluid flows are necessary conditions for successful operation. Significant sources of dispersion are: 1) uneven (parabolic) flows; 2) electrohydrodynamic flows; 3) molecular diffusion; 4) thermal convection; 5) sedimentation; 6) thermally induced sample mobility variations; and 7) electroosmosis.
In continuous zone electrophoresis (CFE), the electrolyte solution flows in a direction perpendicular to the electric field and the mixture to be separated is inserted continuously into the flowing solution. Components of the mixture are deflected according to their electrophoretic mobilities and can be collected continuously after their migration. Svensson and Brattsten were the first to report a method for carrying out electrophoresis continuously. They used a lateral electric field in a narrow plexiglas box packed with glass powder as an anti-convective medium. Durrum modified the above configuration by replacing the glass-filled box with a filter paper curtain, hanging in a free vapor space. While both of these methods demonstrated continuous electrophoresis, they both used a stabilizing medium. Anti-convective media cause many problems such as reduction of the flow capacity by their presence, electroosmosis in the interstices, adsorption of the sample and xe2x80x9cpacking or eddy diffusionxe2x80x9d. Efforts were then made to do continuous electrophoresis in a free fluid. Bier in 1957 reported the first continuous flow electrophoresis device which could separate two protein solutions by adjusting the buffer pH relative to the isoelectric point of one of the solutions. The device which he described as xe2x80x9ccontinuous free-boundary flow electrophoresisxe2x80x9d did not take place in a single rectangular chamber and did not produce a separation of high purity. Dobry and Finn (U.S. Pat. No. 3,149,060) were the first to report continuous flow free fluid electrophoresis in a rectangular chamber with a cross-section of low aspect ratio, hence providing little resistance to thermal convective flow disturbances. This configuration was limited to very low electric fields and required the use of buffer thickening agents to suppress convective eddies. Philpot described a continuous flow electrophoresis system with the electric field applied across (perpendicular to) a thin film of liquid. He later wrapped his thin film geometry into a thin annulus surrounded by two concentric cylinders (electrodes). The outer cylinder rotated to provide a stabilizing velocity gradient.
Although a large throughput, 10 g/hr, was accomplished by the Biostream, its resolution was poor. This was followed by forced flow electrophoresis devised by Bier for the large scale purification of a single component in a mixture. Giddings extended this development with field flow fractionation wherein an electric field has been just one example of the force field deflecting the sample across the narrow plane. The need for flat, uniform surfaces that also serve to isolate the electrode arrays have slowed this development. Mel in 1959 reported the first use of a high aspect ratio rectangular separation chamber using a lateral electric field. The xe2x80x9cthinxe2x80x9d chamber of 0.7 cm thickness provided the necessary wall interaction to suppress thermal convective flows to the extent that a less viscous free flow buffer could be used. This design served as the impetus for the development of the conventional CFE machines of the 60""s and 70""s with their chamber cross-section of high aspect ratio and laterally directed electric fields. During this time frame, Hannig and his co-workers developed CFE by making the chamber cross-sections even thinner, approaching 0.25 cm for some designs. Unfortunately, the gains made in suppressing thermal convection were wiped out by electrohydrodynamic interaction with intrinsic chamber fluid flows to cause crescent-shaped distortions. Nevertheless, a variety of CFE instruments were manufactured according to the designs of Hannig (in Germany) and Strickler (in the US) (U.S. Pat. No. 3,412,008) and several hundred instruments were used in laboratories around the world. Rhodes and Snyder subsequently devised a technique to minimize these flow distortions (U.S. Pat. No. 4,752,372).
The concept of counterflow to oppose the electrophoretic migration was first described to the inventors by Griffin and McCreight as a means to attenuate the crescent shaped distortion in CFE chambers. Richman subsequently patented a similar counter-flow method where axial bands of electroosmotic coatings of varying zeta potential would xe2x80x9cstraightenxe2x80x9d distorted sample bands (U.S. Pat. No. 4,309,268). The method was impractical because most coatings change with time and there exists no spectrum of coatings with respect to zeta potential. A more practical approach that did not use counter-flow was suggested by Strickler wherein the CFE was divided into two vertical compartments, each with a different wall coating, so that the combined electroosmotic flow would yield a more coherent sample band. Subsequently, Ivory used counter-flow to increase sample residence time in a recycling CFE. Egen, et al. have also devised a counterflow gradient focusing method (U.S. Pat. No. 5,336,387).
While the crescent phenomenon was long known to cause untenable sample stream distortion in CFE instruments, it was not until 1989 that Rhodes and Snyder showed that electrohydrodynamics transforms initially circular sample streams into ribbons that initiate the crescent shaped distortions. The operation of CFE devices was labor intensive and unreliable due to contamination of the closely spaced chamber walls and the resultant electroosmotic flow variations through the chamber.
Isoelectric focusing (IEF) is an electrophoretic technique that adds a pH gradient to the buffer solution and together with the electric field focuses most biological materials that are amphoteric. Amphoteric biomaterials such as proteins, peptides, nucleic acids, viruses, and some living cells are positively charged in acidic media and negatively charged in basic media. During IEF, these materials migrate in the pre-established pH gradient to their isoelectric point where they have no net charge and form stable, narrow zones. Isoelectric focusing yields such high resolution bands because any amphoteric biomaterial which moves away from its isoelectric point due to diffusion or fluid movement will be returned by the combined action of the pH gradient and electric field. The focusing process thus purifies and concentrates sample into bands that are relatively stable. This is a powerful concept that has yielded some of the highest resolution separations, especially when coupled with electrophoresis in two-dimensional gels. Unfortunately there are drawbacks to IEF that have limited its applications. The rate of electrophoretic migration of each charged species decreases progressively as it approaches its isoelectric point and long residence times are required for high resolution. Proteins have reduced solubility at their isoelectric point although precipitation of the concentrated bands can be minimized by addition of detergent. Additional problems relate to the commercial amphoteric solutions, including: 1) difficulty of extracting the separated proteins, peptides, etc., from the amphoteric solutions because of their similar physical properties and interactions; 2) chemical toxicity; 3) handling problems; and 4) cost.
IEF had its practical beginning in the mid-1950""s when Kolin first demonstrated the concept of focusing ions in a pH gradient by placing a molecular sample between an acidic and a basic buffer and applying an electric field. Although the constituents focused rapidly, the gradient soon deteriorated due to the concurrent electrophoretic migration of all of the buffering ions. The synthesis of stable carrier ampholytes by Vesterberg and their successful commercial development led to broad use in gels or other restrictive media to suppress electroosmosis and thermal convection during analytical separations.
The high resolution achieved by IEF encouraged many attempts to develop a preparative version of the process. This proved to be much more difficult for IEF than zone electrophoresis because of the variable fluid properties and sample characteristics within the chamber leading to changing values of electroosmosis and thermal convection during the separation. Various CFE devices were modified to run with an amphoteric mixture instead of buffer but the problems (long focusing time requiring a slow flow through the chamber, pH drift toward the cathode, reduced voltage/current levels for acceptable heating and convection) became insurmountable. A. J. P. Martin described a means of performing large-scale isoelectric focusing by connecting a number of separation chamber in series via membranes. By circulating the fluids in each compartment through external coolers, Martin claimed that the removal of heat had been solved. Since the only pH shift occurred across the membranes, the pH gradient was quite steep between chambers. Bier further developed the external cooling system, added sensors and demonstrated the improved focusing with recycling (U.S. Pat. No. 4,362,612). Bier added a stabilizing assembly rotation to the membrane segmentation and a novel collection system (U.S. Pat. No. 4,588,492) which led to the Roto-Phor from Bio-Rad (Hercules, Calif.). Righetti has also extended the multi-compartment concept by using membranes, cast and polymerized with the desired amphoteric molecules inside, to establish the pH gradient rather than preparing a constant pH in each compartment. The Iso-Prime system (Hoefer Instruments, San Francisco, Calif.) is based upon a stack of membranes with buffer between them. The pH gradient develops rapidly and the proteins move through the membranes until they reach the cell with the pH equal to their isoelectric point. Although the membranes stabilize the focusing process, they become clogged if the protein precipitates in them.
Thus, prior methods of isoelectric focusing have suffered from the many drawbacks outlined above, and have also been hindered by problems during the transition from an analytical system to a preparative system that have limited its intended use. It is thus highly desirable to develop a focusing system for separating biological molecules and other components in a mixture which is able to avoid all of the problems of the prior art and which can achieve high resolution of separation in an analytical or a preparative mode through a practically unlimited scale-up potential. It is also highly desirable to develop an electrophoretic focusing system which can control the adverse effects of Joule heating and electrohydrodynamics on the electrophoretic separation procedure.
It is an object of the present invention to provide a preparative-scale free-fluid electrophoretic separator with high resolution as well as an analytical capability commensurate with capillary zone electrophoresis. The particular mode of high-resolution separation as provided by the present invention, which is referred to as electrophoretic focusing, combines features of electrophoresis and isoelectric focusing to accomplish large scale purifications and fractionations that have not been possible before now.
It is another object of the present invention to develop a separation device capable of high speed and short residency through the use of high voltage gradients. These high voltage gradients are produced by relatively low voltages applied across the narrow chamber dimensions. Another object is flexibility with operation in either a constant electric field, continuous flow mode or in a linearly varying electric field batch mode. Both modes permit scanning of the sample fraction content and display in a conventional histogram format. The goal of high resolution of separation can be achieved through the use of the present invention in an analytical or a preparative mode through a practically unlimited scale-up potential. A further goal is to control the adverse effects of Joule heating and electrohydrodynamics.
These and other objects and benefits are achieved by the use of the present invention which provides a number of innovations and insights with regard to fundamental fluid and thermal geometries and operations. The focusing is accomplished with a minimum of sample migration which leads to a higher resolution in a shorter time. Adiabatic thermal conditions in the lateral (scale-up) dimension permit a large increase in throughput at no apparent loss of resolution. Active cooling limits the maximum chamber temperature and its relationship to the chamber orientation and buffer fluid transport is such as to limit thermal convection. Porous, rigid screens permit a controlled focusing cross-flow which balances the electrophoretically-driven sample velocity.